The present invention relates to a composition comprising a plurality of polynucleotide probes for use in research and diagnostic applications.
DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer. The array has over 50,000 DNA probes to analyze more than 400 distinct mutations of p53. A cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drug metabolism, drug resistance or drug toxicity.
DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes. There is a growing awareness that gene expression is affected in a global fashion. A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes. In some cases the interactions may be expected, such as where the genes are part of the same signaling pathway. In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.
cDNA-based arrays have been used in discovery and analysis of inflammatory disease related genes (Heller et al. (1997) Proc. Natl. Acad. Sci USA 94: 2150-2155). A first type of array was employed to characterize the expression patterns of a class of 96 genes coding for polypeptides known to be involved in rheumatoid arthritis. This array contained preselected probes for the 96 genes. A second type of array was used to investigate gene expression patterns characteristic of blood cells. This array contained probes for 1,000 human genes randomly selected from a human blood cell cDNA library.
Current cDNA-based arrays suffer from a variety of limitations. One is the first type of array can only detect the expression patterns of a limited number of genes already associated with a disease. The expression of other, yet to be identified, relevant genes is not detected. Another is the second type of array contains probes for genes that have very little to do with the regulation of inflammation. Also, high abundance genes are likely to be over represented and low abundance genes are likely to be under represented. The present invention provides a way to overcome such limitations.
In one aspect, the present invention provides a composition comprising a plurality of polynucleotide probes, wherein each of said polynucleotide probes comprises at least a portion of a gene implicated in blood cell biology. The plurality of polynucleotide probes can be selected from I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology; and IV) combinations of first, second or third polynucleotide probes.
Preferably, the plurality of polynucleotide probes comprises: I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; and III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology.
Generally, first polynucleotide probes are selected by a) preparing at least one first target transcript profile from a first biological sample selected from the group consisting of hematopoietic cells and inflamed tissue and at least one first subtraction transcript profile from a noninflamed, nonhematopoietic biological sample; b) subtracting said first subtraction transcript profile from said first target profile to detect a plurality of genes that are differentially expressed in an immunological response; and c) identifying one of said detected genes that are differentially expressed in an immunological response. Second polynucleotide probes are selected by a) preparing at least one second target transcript profile from a second biological sample selected from the group consisting of hematopoietic cells and inflamed tissue to detect genes that are abundantly expressed in said second biological sample; and b) identifying one of said detected genes that are abundantly expressed. Third polynucleotide probes are selected by a third method comprising identifying a gene coding for a polypeptide with a known function in immunological responses.
In one preferred embodiment, the composition comprises a plurality of polynucleotide probes, wherein each polynucleotide probe comprises at least a portion of a sequence selected from the group consisting of SEQ ID Nos: 1-1508. In a second preferred embodiment, the composition comprises a plurality of polynucleotide probes comprising at least a portion of at least about 1000 of the sequences of SEQ ID Nos: 1-1508. In yet another embodiment, the composition comprises a plurality of polynucleotide probes wherein said polynucleotide probes comprise at least a portion of substantially all the sequences of SEQ ID Nos: 1-1508. The polynucleotide probes can be cDNAs, clone DNAs and the like.
The composition is particularly useful as hybridizable array elements in a microarray for monitoring the expression of a plurality of target polynucleotides. The microarray comprises a substrate and the hybridizable array elements. The microarray can be used, for example, in the diagnosis and treatment of an immunopathology.
In another aspect, the present invention provides an expression profile that can reflect the levels of a plurality of target polynucleotides in a sample. The expression profile comprises a microarray and a plurality of detectable complexes. Each detectable complex is formed by hybridization of at least one of said target polynucletodies to at least one of said polynucleotide probes and further comprises a labeling moiety for detection.
In yet another aspect, the present invention provides a method for identifying a plurality of polynucleotide probes. The method comprises selecting I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; and III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology.